Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (10): 1930-1942.doi: 10.3864/j.issn.0578-1752.2024.10.007

• PLANT PROTECTION • Previous Articles     Next Articles

Function and Mechanism Analysis of Vm-milRN7 Regulating the Pathogenicity of Valsa mali

ZHANG Jian(), ZHAO BinSen, FENG Hao(), HUANG LiLi()   

  1. College of Plant Protection, Northwest A&F University/State Key Laboratory for Crop Stress Resistance and High-Efficiency Production, Yangling 712100, Shaanxi
  • Received:2023-12-18 Accepted:2024-02-16 Online:2024-05-16 Published:2024-05-23
  • Contact: FENG Hao, HUANG LiLi

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Abstract:

【Background】MicroRNA-like RNAs (milRNAs) are a class of regulatory factors commonly found in fungi with similar generation and action mechanism as plant and animal microRNAs, which are widely involved in their growth and development, as well as in life activities such as infection and pathogenesis of plant pathogenic fungi. The apple Valsa canker caused by Valsa mali is the most destructive disease affecting apple production.【Objective】 The research aims to explore the function and mechanism of Vm-milRN7 in regulating the pathogenicity of V. mali, and to provide a theoretical basis for targeted disease resistance breeding of apple Valsa canker.【Method】 Vm-milRN7 precursor overexpression vector was constructed by amplifying Vm-milRN7 precursor sequence using genomic DNA of strain 03-8 as a template; upstream and downstream sequences of Vm-milRN7 precursor were amplified and Vm-milRN7 precursor knockout mutants were constructed by using Double-joint PCR technique. The Vm-milRN7 precursor overexpression strains and knockout mutants were constructed by PEG-mediated protoplast transformation. The vegetative growth rate of Vm-milRN7 precursor overexpression strains and knockout mutants was determined by cultivation on PDA medium, and the pathogenicity of these strains was verified by inoculation on apple twigs and leaves. The regulatory relationship between Vm-milRN7 and its potential target gene Vm-09496 was identified by qRT-PCR and co-infiltration experiment in Nicotiana benthamiana leaves; protein sequence characterization and phylogenetic analysis of Vm-09496 were performed using bioinformatics software. In order to analyze its function, Vm-09496 knockout mutants and complement strains were created and their phenotypes were characterized.【Result】 PEG-mediated genetic transformation was used to create Vm-milRN7 overexpression strains and knockout mutants. Vm-milRN7 overexpression strains showed no apparent alteration in vegetative growth rate compared with that of the wild-type strains, whereas the knockout mutants had a significantly lower vegetative growth rate. Compared with the wild-type strains, the overexpression strains showed a significant increase in the pathogenicity of V. mali on apple leaves, while the knockout mutants showed a significant decrease in the pathogenicity of V. mali on apple leaves and twigs. Further, qRT-PCR and co-infiltration in N. benthamiana leaves assay showed that Vm-milRN7 inhibited the expression of its potential target gene Vm-09496. Bioinformatics analysis revealed that the gene encodes an imaginary protein and is evolutionarily most closely related to VP1G_09956 in V. pyri. In order to analyze its function, the Vm-09496 knockout mutants and complement strains were created, and the pathogenicity of the knockout mutants on both twigs and leaves was significantly increased compared with that of the wild-type strain, while the vegetative growth rate and pathogenicity of the complement strains recovered to the wild-type level.【Conclusion】 Vm-milRN7 may be involved in the pathogenity of V. mali by regulating the expression of the target gene Vm-09496. The target gene Vm-09496 is an important endogenous gene affecting V. mali infection and negatively regulates V. mali pathogenicity.

Key words: Valsa mali, milRNA, knockout mutant, pathogenicity, post-transcriptional regulation

Table 1

Primers used in this study"

引物名称Primer name 引物序列Primer sequence (5′-3′)
Vm-milRN7OE-F GTAGGAACCCAATCTTCAAATTCGTCTCCAAGCA
Vm-milRN7OE-R TGAATGTTGAGTGGAATGATAGCAGTTCCAATTC
Vm-milRN7-1F GACCGTATCGGTATCACCTAACG
Vm-milRN7-2R CAGATACGGCAGAGAAATCGCAACCTCACAGCAAATCTCACGACCACTTC
Vm-milRN7-3F GTTTAGATTCCAAGTGTCTACTGCTGGCCAGGATCGACAGACTGAACAGAA
Vm-milRN7-4R CTAGAGGAATAACGAGGAACGGA
Vm-milRN7-5F CCGCCCAAGTATTTCCGACC
Vm-milRN7-6R AAACCTCTCCGCCGCCTAGA
Vm-milRN7-7F AGTGGAGGCATAGCAATCAATT
Vm-milRN7-8R TTCAACAGGTCCAAGGTTTAGG
Vm-09496-1F CTGCTTGGCATGTTCCCTTAC
Vm-09496-2R CAGATACGGCAGAGAAATCGCAACCTCCCCGCTTTGCTTACTTTGTGT
Vm-09496-3F GTTTAGATTCCAAGTGTCTACTGCTGGCGGACCACATCACTACCTACTCG
Vm-09496-4R TATTGCTCTATCCTGGCTTTCT
Vm-09496-5F CCGATACCGACAGCCTTTACTT
Vm-09496-6R GCTCATGGTCTGAATCTCCACC
Vm-09496-7F TTTGGTGCCACTGTCGTCTATC
Vm-09496-8R AACGGGCTTCTCAATCTCATGT
HB-Vm-09496-F TCTCATCACCATCACCATCACCAGGATTGAAGGTCCCGGTGCG
HB-Vm-09496-R TCGCCCTTGCTCACCCTCGAGGGAAGGGCTTTTTTCATTAGCAGCACA
Pri-N7-F CATTTCATTTGGAGAGGACCATGACACAGTGAAGTGGTCGTGAGATT
Pri-N7-R TCTAGTTCATCTAGAGGATCAAGCAGTTCCAATTCGATGTATTC
Tar-Vm-09496-F CATTTCATTTGGAGAGGACCATGTTCCGCCTCGACTCCCGCCACGTCG
Tar-Vm-09496-R GTTGTGTTGAGAATTCTCGAGGCGGTGCCGAACTCCTGGGCCCCT
RT-Vm-milRN7 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTAAATC
qRT-Vm09496-F TTGTGTTTGGTCTGGTGTTGC
qRT-Vm09496-R CGAGTCTGATTCCGTTTTCGT
G850-F TCGGCTATGACTGGGCACAACA
G852-R GAGCGGCGATACCGTAAAGCAC
G855-R TGTTGGGTTTGAGCTAGGTGGGGTGA
G856-F GAATGGTCAAATCAAACTGCTAGATAT
RT-VmU6 GCTTTCTTTGGTATAGCGTGTCA
VmU6-F GGTCAATTTGAAACAATACAGAGAAGAT
VmU6-R TCTTTGGTATAGCGTGTCATCCTTAG

Fig. 1

Diagram of strategy of generation target gene/sequence deletion constructs"

Fig. 2

Identification of Vm-milRN7 overexpression strains (A) and knockout mutants (B)"

Fig. 3

Phenotype determination of Vm-milRN7 overexpression and WT strains"

Fig. 4

Phenotype determination of Vm-milRN7 knockout mutants and WT strains"

Fig. 5

Protein sequence analysis of the target gene Vm-09496"

Fig. 6

Detection of Vm-09496 expression level in WT, Vm-milRN7 overexpression strains and knockout mutants"

Fig. 7

Analysis about the regulation relationship between Vm-milRN7 and Vm-09496 by co-infiltration in N. benthamiana leaves"

Fig. 8

Identification of Vm-09496 knockout mutants"

Fig. 9

Identification of the gene complement strains of HB-Vm-09496"

Fig. 10

Vegetative growth rates of WT, Vm-09496 knockout mutants and complement strains"

Fig. 11

Pathogenicity of WT, Vm-09496 knockout mutants, and complement strains on leaves and twigs"

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